The present invention relates to proteins which have an enzymatic activity for converting β-carotene into zeaxanthin or canthaxanthin into astaxanthin, to nucleic acids which encode these proteins, to nucleic acid constructs comprising these nucleic acids, to genetically manipulated organisms where the genetic manipulation causes or increases the gene expression of this nucleic acid by comparison with a wild-type, and to processes for preparing xanthophyll derivatives.
Xanthophylls are oxygen-containing carotenoids of animal, plant or microbial origin. Xanthophylls such as lutein, zeaxanthin or astaxanthin are important additives in the human and livestock diet as pigmenting substances and precursors of vitamin A derivatives. In addition, xanthophylls have a health-promoting action such as enhancing the immune response and, by reason of their antioxidant properties, a cancer-preventing action, which makes their use as nutraceuticals of interest. An economic process for preparing xanthophylls and foodstuffs with an increased xanthophyll content are therefore of great importance. Particularly economic processes for preparing xanthophylls are biotechnological processes which make use of proteins and biosynthesis genes of xanthophyll biosynthesis from xanthophyll-producing organisms.
Prokaryotic β-carotene hydroxylases which catalyze the enzymatic conversion of β-carotene into zeaxanthin via β-cryptoxanthin, and the genes which encode these proteins are known from the bacteria Erwinia uredovora (Misawa et al., J. of Bacteriology 1990, 6704–6712; EP 393690 B1), Erwinia herbicola (WO 9113078), Agrobacterium aurantiacum (Misawa et al., J. of Bacteriology 1995, 6575–6584; EP 735 137 A1), Alcaligenes sp. PC-1 (EP 735 137 A1), Flavobacterium sp. strain R1534 (Pasamontes et al., Gene 1997, 185, 35–41; EP 747483 A2) and from the Cyanobacterium Synechocystis sp. PCC6803 (Masamoto et al., Plant Cell Physiol. 1998, 39(5), 560–564).
It is also known that the prokaryotic β-carotene hydroxylases from Agrobacterium aurantiacum, Alcaligenes and Erwinia uredovora are additionally able to covert canthaxanthin via adonirubin in astaxanthin (Misawa et al., J. of Bacteriology 1995, 6575–6584; Fraser et al., J. Biol. Chem. 1997, 272, 6128–6135).
From eukaryotic sources, three plant β-carotene hydroxylases are known to catalyze the enzymatic conversion of β-carotene into zeaxanthin via β-cryptoxanthin. The corresponding cDNAs have been isolated from Arabidopsis thaliana (Cunningham et al, J. Biol. Chem. 1996, 271, 24349–24352, WO 9736998), and from Capsicum an-nuum L. (Bouvier et al., Biochimica et Biophysica Acta 1998, 1391, 320–328).
Genes of eukaryotic origin have the advantage over prokaryotic genes that they are expressed better in higher transgenic organisms such as plants. Nevertheless, there is still a need to improve and increase the xanthophyll productivity for an economic process for preparing xanthophyll derivatives or foodstuffs with an increased xanthophyll content by incorporating eukaryotic nucleic acids into organisms.
In addition, the appropriate eukaryotic β-carotene hydroxylases in the prior art have the disadvantage that they have only a narrow substrate range so that there is a build up of metabolic products which cannot be converted by the hydroxylases and may exert an inhibiting effect on the hydroxylases.